Using WES in distant

relationships to identify

cardiomyopathy genesRobert M. (Bob) Hamilton

Professor and Senior Associate Scientist

The Hospital for Sick Children & Research Institute

Hereditary Cardiomyopathies

• Dilated Cardiomyopathy (dilated LV and i function)

• Ischemic, Inflammatory/Immune, Hereditary (25-50% of DCM)

• Identified Genes in 40% of Hereditary DCM

• Arrhythmogenic Cardiomyopathy

• Ventricular Arrhythmias (predominantly from RV)

• Fibrofatty Myocardial Replacement (predominantly of RV)

• Other:Hypertrophic (~75% gene-ID’ed), Restrictive (rare)

Hershberger et al.

Nat. Rev. 2013

DCM Family 1

• A multiplex family with dilated cardiomyopathy (red) resulting in

cardiac transplantation in 3 (green)

• Oldest generation died without sampling; youngest generation does

has not yet manifest a phenotype; * indicates DNA available

• Thus, traditional segregation analysis will not inform gene discovery

BCL2-associated athanogene 3 (BAG3)

Case series of CJET: Familial Occurences

• Villain and colleagues (1990): 26 subjects

• 5 sib-pairs (4 non-identical twin pairs)

• 1 cousin relationship

• 1 subject’s father had accelerated

• Collins and colleagues (2009): 99 subjects

• familial association in 20%

Congenital Junctional Ectopic Tachycardia

(Lancaster Amish Family)

• Kevin Strauss and Eric Puffenberger

(Clinic for Special Children, Lancaster PA)

• SNP mapping identified a putative locus

• WES (Broad Institute) has not identified a candidate

to date

Congenital Junctional Ectopic

Tachycardia (Upstate NY Family)

• Dr. Jeff Vinocur, Golisano Children’s Hospital, Rochester

• Extended family drove to Toronto as research volunteers

• WES performed on 3/17 samples: 3 candidate genes

• Sanger sequencing to confirm and expand segregation

Congenital Junctional Ectopic

Tachycardia Gene Candidates

Congenital Junctional Ectopic Tachycardia

Online 11 March 2015

Causal gene mutation• TNNI3K: c.1729C>T; p.Leu577Phe

• Gene had just previously been associated with CJET and familial conduction system disease

(Xi et al., Int J Cardiol 2015)

TNNI3K

• Troponin I-interacting kinase

• Kinase with cardiac-restricted expression

• Has been implicated in various cardiac phenotypes

and diseases including heart failure,

cardiomyopathy, ischemia/reperfusion injury and

conduction of the cardiac electrical impulse

Mutation carrier

Non mutation carrier

Family screening

Functional study• The identified gene mutation is inserted into a

zebrafish model (using CRISPR/Cas gene editing)

• Zebrafish will be assessed for tachyarrhythmia

Basic DNA editing using

CRISPR/Cas9

From: The Jackson laboratory

Zebrafish model

• Gain or loss of function?

• Screen small molecule libraries focused on TNNI3K

inhibitors (or alternately kinase activators) to

influence this pathway involved in

ischemia/reperfusion injury, oxidative stress, and

myocyte death

• Assessment of FDA-approved generic drugs

Contributors• University of Rochester:

• Jeffrey Vinocur

• The Hospital for Sick Children:

• Tamara Koopmann

• Meena Fatah

• Sarah Hutchinson

• James Dowling

• Robert Hamilton

robert.hamilton@sickkids.ca

Supported by the

Paul C. Gillette fund

Ped. & Cong. EP Soc.

Gene Discovery in

Arrhythmogenic Cardiomyopathy 1

• “Although the QT prolonged from 25 ± 1.0 to 30 ± 1.1 ms in mice, the difference could be due to delayed conduction”

• “PR intervals increased from 35.9 ± 1.0 to 39.6 ± 0.7 ms, QRS intervals increased from 8.3 ± 0.1 to 11.2 ± 0.2 ms, and P-wave duration increased from 8.2 ± 0.7 to 13.4 ± 0.5 ms for wild-type v. mutant mice; all differences statistically significant, P < 0.05)”

• “Given that action potentials of adult mutant cardiomyocytes are not substantially prolonged, the increase in QT interval observed in mice is probably due to delayed conduction”

Gene Discovery in

Arrhythmogenic Cardiomyopathy 2

WT Mutant

Embryonic Mouse

Heart

Gene Discovery in Canine

Arrhythmogenic Cardiomyopathy• Associated with Striatin 3’UTR mutation in American dogs

• Does not segregate among Canadian, UK or European dogs

Golden Helix SVS• Provides for facile analysis of SNP, Exome & Genome

data to users without major bioinformatic background

• Relatively easy to learn

• Things I like:

• Many analyses in one program (Swiss Army Knife)

• Rapid implementation of new reference data

• Integration with & free BAM viewer

• Amazing company support

Golden Helix SVS

• Examples of company support (things I asked for)

• Multiplex pedigree function (Greta suggested importing

pedigree file and then assigning genotype data)

• Demonstration of protein-level effects (Greta showed me

how to apply feature labels from protein coding field)

• User-friendly tools for file conversions: (FASTA/Q > SAM

> BAM > VCF) (Offered to perform file conversions)

• Implement Estimation of Recent Shared Ancestry

(Offered to look at algorithms